Bottom Line:
All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol.The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient.The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient.

Affiliation: Lankenau Institute for Medical Research, Wynnewood, PA, 19096, United States of America.

ABSTRACTThe micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed.

pone.0133926.g003: Change of tight junctional proteins as a function of state of differentiation of the CACO-2 cell layer.The relative changes in the abundance of eight tight junctional proteins occurring as a function of days post confluence of the cell layer. 3-day, 7-day, and 21-day post-confluent CACO-2 cell layers in Falcon 75 cm2 culture flasks, having been refed with control medium at confluence and every 2–3 days thereafter with control medium, were harvested in lysis buffer. Further steps were performed as described in Table 1. Data represents the percent of 3-day cell layers, and is expressed as the mean ± standard error for an n = 3 cell layers in all cases. NS indicates non significance. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001 (one-way ANOVA followed by Dunnett’s post hoc testing versus day 3).

Mentions:
We observed that the state of differentiation of the cell layer is an important determinant of the response of the cell layer’s barrier integrity to micronutrients. This has not only biomedical significance but also procedural importance concerning the exact manner in which one performs these studies. It is well known that the CACO-2 cell layer slowly differentiates over time once the culture achieves confluence, and that as many as 21 days (post-confluence) can be needed for certain differentiated properties to appear [30]. In Fig 3, we show that TJ proteins are no different in this respect, with changes in the relative abundance of these proteins occurring as a function of days post-confluence. For cells seeded onto Millicell PCF membranes, this equates to days post-seeding, as cells are seeded into PCF units at a confluent cell density. For most of the TJ proteins, a graded increase in abundance was observed (claudins -1, -4, -5, -7 and tricellulin), but with certain noteworthy exceptions. Claudin-2 abundance dramatically plummets by day 21. On the other hand, claudin-3 levels showed absolutely no change as a function of days post-confluence of the cell layer. Interestingly, a significant decrease in occludin levels was also observed by 21 days. In summary, the TJ complex appears to be changing as the cell layer differentiates. This is seen in a functional manner in Table 2, where by 7 days, a maximal elevation of Rt and decrease in mannitol permeability (Jm) is seen. Further improvement in these two parameters of barrier integrity is not evident after day 7, although, as seen in Fig 3, dramatic changes in overall abundance of TJ proteins are still ongoing. Differentiation of the cell layer is still continuing after day 7, as shown by the graded significant increases in Isc from 3 to 7 to 21 days (Table 2).

pone.0133926.g003: Change of tight junctional proteins as a function of state of differentiation of the CACO-2 cell layer.The relative changes in the abundance of eight tight junctional proteins occurring as a function of days post confluence of the cell layer. 3-day, 7-day, and 21-day post-confluent CACO-2 cell layers in Falcon 75 cm2 culture flasks, having been refed with control medium at confluence and every 2–3 days thereafter with control medium, were harvested in lysis buffer. Further steps were performed as described in Table 1. Data represents the percent of 3-day cell layers, and is expressed as the mean ± standard error for an n = 3 cell layers in all cases. NS indicates non significance. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001 (one-way ANOVA followed by Dunnett’s post hoc testing versus day 3).

Mentions:
We observed that the state of differentiation of the cell layer is an important determinant of the response of the cell layer’s barrier integrity to micronutrients. This has not only biomedical significance but also procedural importance concerning the exact manner in which one performs these studies. It is well known that the CACO-2 cell layer slowly differentiates over time once the culture achieves confluence, and that as many as 21 days (post-confluence) can be needed for certain differentiated properties to appear [30]. In Fig 3, we show that TJ proteins are no different in this respect, with changes in the relative abundance of these proteins occurring as a function of days post-confluence. For cells seeded onto Millicell PCF membranes, this equates to days post-seeding, as cells are seeded into PCF units at a confluent cell density. For most of the TJ proteins, a graded increase in abundance was observed (claudins -1, -4, -5, -7 and tricellulin), but with certain noteworthy exceptions. Claudin-2 abundance dramatically plummets by day 21. On the other hand, claudin-3 levels showed absolutely no change as a function of days post-confluence of the cell layer. Interestingly, a significant decrease in occludin levels was also observed by 21 days. In summary, the TJ complex appears to be changing as the cell layer differentiates. This is seen in a functional manner in Table 2, where by 7 days, a maximal elevation of Rt and decrease in mannitol permeability (Jm) is seen. Further improvement in these two parameters of barrier integrity is not evident after day 7, although, as seen in Fig 3, dramatic changes in overall abundance of TJ proteins are still ongoing. Differentiation of the cell layer is still continuing after day 7, as shown by the graded significant increases in Isc from 3 to 7 to 21 days (Table 2).

Bottom Line:
All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol.The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient.The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient.

Affiliation:
Lankenau Institute for Medical Research, Wynnewood, PA, 19096, United States of America.

ABSTRACTThe micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed.